R Notebook

Population Structure

Goal: to determine pop structure from VCF files.

Input files:

• We assume that all genotypes are stored as a VCF (see variant call format specifications: https://samtools.github.io/hts-specs/VCFv4.2.pdf), or BCF (binary VCF) file.
  
• There is no need to pre-qc these genotypes. This workflow will filter on minor allele frequency and call frequency. If you also want to filter on Hardy-Weinberg equilibrium, feel free to adjust as required.

Output:

• There are 2 key output files for this workflow:
  • population_structure.eigenvec: contains the principal components, i.e. the genotypes after rotation. These can be plotted (e.g. PC1 vs. PC2) to visualise population-levels trends in the dataset
    
  • population_structure.eigenval: contains the eigenvalues from the principal components analysis, i.e. the amount of variation explained by each principal component.

Population Structure Workflow:

• filter the genotypes, removing pairs of SNPs which are in high linkage disequilibrium (LD) with each other.
  
• QC the genotypes & perform principal components analysis via plink
  
• produce scree plot of eigenvalues
  
• optionally, produce plots of the population structure

Setup

The workflow assumes that you are using plink2 (aka plink 1.9, available here: https://www.cog-genomics.org/plink2). This has been tested on Ubuntu 16.04. It will require tweaking to run on Windows machines.

Please review the setup chunk below and make sure to set the root.dir option. This directory will become the working directory where all intermediates outputs are stored, so think about a sensible place for this (i.e. prob some scratch directory rather than a Git repo).

Below:

• declare you plink directory
  
• specify the directory where your genotype files are stored
  
• specify the working directory (all intermediate and final outputs will be placed here)
  
• review the workflow parameters, and change if necessary
  
• then, run the cell (will setup parameters, test plink installation and load necessary R packgaes)

NOTE: that I have set the working_directory variable the same as the root.dir option in the setup chunk.

plink_ <- "/mnt/DataDrive/MedicagoFiles/Analyses/Pipeline/PLINK/plink"
genotype_directory <- "/mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload/"
working_directory <- "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/"
# Genotype QC settings
parameters <- list(
    call_freq = 0.10,                       # rejects SNPs with more than this number of missing calls
    maf = 0.05,                             # rejects SNPs with a MAF < this threshold
    pop_struct_dimensions = 10,             # the number of principal components for population structure
    file_format = "bcf"
)
# test plink installation
if (!capture.output({ cat(system(sprintf("%s --version", plink_))) }) == 0) {
    cat(sprintf("
        --------------------------------------------------------------------------------
                                      !!!! WARNING !!!! 
        PLINK not found. Please review the plink_ directory in the Setup section.
        --------------------------------------------------------------------------------
    ", plink_))
}

PLINK v1.90b3.31 64-bit (3 Feb 2016)

# install & load packages
options(repos = "http://cran.uk.r-project.org")
if (!require(data.table)) {    
    install.packages("data.table")
}
if (!require(ggplot2)) {
    install.packages("ggplot2")
}
library(ggplot2)
library(data.table)

Genotype QC and data conversion

Plink can accept VCF and BCF files directly as inputs. However, in our case the Medicago BCF files do not have SNP identifiers (annoying - yes!), necessary for the LD pruning step. Below, we will perform the genotype QC and output plink-formatted (bed, bim and fam) files. For more information about plink formats please refer to: https://www.cog-genomics.org/plink2/formats#bed

The plink command is annotated below for reference:

plink --bcf <genotype_file> \\                     # points to a BCF file
        --const-fid \\                             # the Medicago data has no family structure, so we ste FID = 0 for all (this is a plink-ism)  
          --allow-extra-chr \\                     # fudge for the Medicago data
          --geno 0.1 \\                            # call frequency threshold
          --maf 0.05 \\                            # MAF threshold
          --make-bed \\                            # create plink-formatted bed, bim, fam files
          --set-missing-var-ids @:#[b37] \\        # create unique SNP ids (refer:https://www.cog-genomics.org/plink2/data#set_missing_var_ids)
          --out <output_file>                      # the name of the output file

Run the cell below. No changes necessary.

This cell will read in all files in the gentoype_directory, which end in the specified file format (in our Medicago example, the filenames must end in “bcf”). For each of these files, it will QC the genotypes and write out plink formatted files to working_directory/plink_genotype_files/.

Review the cell below and run (no changes necessary).

genotype_files <- list.files(genotype_directory, pattern = sprintf("%s$", parameters$file_format), full.names = TRUE)
output_directory <- sprintf("%splink_genotype_files/", working_directory)
if (!dir.exists(output_directory)) {
    dir.create(file.path(output_directory))
}
for (genotype in genotype_files) {
    
    fileparts <- strsplit(genotype, "/")[[1]]
    filename <- strsplit(fileparts[length(fileparts)], "\\.")[[1]][1]
    
    cmd <- sprintf("
        %s --%s %s \\
           --const-fid \\
           --allow-extra-chr \\
           --geno %s \\
           --maf %s \\
           --make-bed \\
           --set-missing-var-ids @:#[b37] \\
           --out %s
    
    ", plink_, parameters$file_format, genotype, parameters$call_freq, parameters$maf,
       sprintf("%s%s", output_directory, filename))
    system(cmd)
    
    
}

PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr1-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15-temporary.fam
written.
4978489 variants loaded from .bim file.
4978489 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.777102.
3311387 variants removed due to missing genotype data (--geno).
1478345 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
188757 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr2-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

15488 MB RAM detected; reserving 7744 MB for main workspace.

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15-temporary.fam
written.
4331584 variants loaded from .bim file.
4331584 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.765386.
2960921 variants removed due to missing genotype data (--geno).
1201379 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
169284 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr3-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15-temporary.fam
written.
5424868 variants loaded from .bim file.
5424868 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.750934.
3799229 variants removed due to missing genotype data (--geno).
1421896 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
203743 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15.log.
Options in effect:
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  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15
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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15-temporary.fam
written.
5414544 variants loaded from .bim file.
5414544 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.772367.
3637500 variants removed due to missing genotype data (--geno).
1569441 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
207603 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15.bim
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PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15.log.
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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15-temporary.fam
written.
4496375 variants loaded from .bim file.
4496375 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.751541.
3177191 variants removed due to missing genotype data (--geno).
1151620 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
167564 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr6-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

15488 MB RAM detected; reserving 7744 MB for main workspace.

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/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15-temporary.bim
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/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15-temporary.fam
written.
3650081 variants loaded from .bim file.
3650081 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.710922.
2801570 variants removed due to missing genotype data (--geno).
719426 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
129085 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr7-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15-temporary.fam
written.
4761519 variants loaded from .bim file.
4761519 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.755777.
3308978 variants removed due to missing genotype data (--geno).
1269547 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
182994 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chr8-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

15488 MB RAM detected; reserving 7744 MB for main workspace.

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15-temporary.fam
written.
4377934 variants loaded from .bim file.
4377934 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.755055.
3038214 variants removed due to missing genotype data (--geno).
1177659 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
162061 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15.log.
Options in effect:
  --allow-extra-chr
  --bcf /mnt/DataDrive/MedicagoFiles/Genotypes/PublicDownload//chru-filtered-set-2014Apr15.bcf
  --const-fid
  --geno 0.1
  --maf 0.05
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15
  --set-missing-var-ids @:#[b37]

15488 MB RAM detected; reserving 7744 MB for main workspace.

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--bcf:
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15-temporary.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15-temporary.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15-temporary.fam
written.
2630449 variants loaded from .bim file.
2630449 missing IDs set.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.698939.
1919960 variants removed due to missing genotype data (--geno).
604512 variants removed due to minor allele threshold(s)
(--maf/--max-maf/--mac/--max-mac).
105977 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15.fam
... 0%0%1%1%2%2%3%3%4%4%5%5%6%6%7%7%8%8%9%9%10%10%11%11%12%12%13%13%14%14%15%15%16%16%17%17%18%18%19%19%20%20%21%21%22%22%23%23%24%24%25%25%26%26%27%27%28%28%29%29%30%30%31%31%32%32%33%33%34%34%35%35%36%36%37%37%38%38%39%39%40%40%41%41%42%42%43%43%44%44%45%45%46%46%47%47%48%48%49%49%50%50%51%51%52%52%53%53%54%54%55%55%56%56%57%57%58%58%59%59%60%60%61%61%62%62%63%63%64%64%65%65%66%66%67%67%68%68%69%69%70%70%71%71%72%72%73%73%74%74%75%75%76%76%77%77%78%78%79%79%80%80%81%81%82%82%83%83%84%84%85%85%86%86%87%87%88%88%89%89%90%90%91%91%92%92%93%93%94%94%95%95%96%96%97%97%98%98%99%done.

The outputted plink files will have substantially less SNPs than the raw genotype files. The exact number of SNPs will be a reflection of the quality of the genotype calls.

LD Pruning

For each plink genotype file, we will further reduce the number of SNPs based on pairwise LD. Li et al (2012, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995003/) provide quite a nice overview of this process. In particular, the methods section states:

“To reduce the LD between SNPs, PLINK was used to conduct SNP-pruning: we considered a sliding window of 1500 SNPs and calculated r2 between each pair of SNPs in that window; one of a pair of SNPs was removed if the pairwise r2 was greater than 0.2, then the window shifted 100 SNPs forward and the procedure was repeated. We used three-dimensional scatter plots of the top 3 significant principal components inferred by SMARTPCA to visualize population structure.” (Li et al, 2012).

We are not going to be quite as stringent as this. We will consider a sliding window of 10 kilobases, with a slide of 10 kilobases and R2 threshold of 0.5.

The following tutorial (http://www.jurgott.org/suzanne2012-07-23/GenABELexercise.pdf) (Wang & Leal, 2012) also has some nice notes with regards to determining population structure. In particular, the authors note that “for PCA and MDS you do not want to use markers which are in LD with each other. Here we only use a subset of GWAS markers for analysis of an actual GWAS data set would want to select a subset of markers which are not in LD” (p.8, Wang & Leal, 2012). This makes good sense, if you consider principal components analysis as a partitioning of covariance.

Review the cell below and run (no changes necessary).

genotype_directory <- output_directory
genotype_files <- unlist(lapply(list.files(genotype_directory, pattern = ".bed$", full.names = FALSE),
                                function (x) strsplit(x, "\\.")[[1]][1]))
for (genotype in genotype_files) {
    cmd <- sprintf("
               
        %s --bfile %s \\
           --indep-pairwise 10 10 0.5 \\
           --out %s
    ", plink_, sprintf("%s%s", genotype_directory, genotype), sprintf("%s%s_pruned_snps", working_directory, genotype))
    
    system(cmd)   
}

PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr1-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr1-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
188757 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr1-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.938265.
188757 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 66234 variants from chromosome 1, leaving 122523.
Pruning complete.  66234 of 188757 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr1-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr1-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr2-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr2-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
169284 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr2-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937878.
169284 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 60256 variants from chromosome 2, leaving 109028.
Pruning complete.  60256 of 169284 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr2-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr2-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr3-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr3-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
203743 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr3-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937249.
203743 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 68199 variants from chromosome 3, leaving 135544.
Pruning complete.  68199 of 203743 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr3-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr3-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr4-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr4-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
207603 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr4-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.93759.
207603 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 72586 variants from chromosome 4, leaving 135017.
Pruning complete.  72586 of 207603 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr4-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr4-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr5-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr5-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
167564 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr5-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937175.
167564 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 59222 variants from chromosome 5, leaving 108342.
Pruning complete.  59222 of 167564 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr5-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr5-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr6-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr6-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
129085 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr6-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.935829.
129085 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 44137 variants from chromosome 6, leaving 84948.
Pruning complete.  44137 of 129085 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr6-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr6-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr7-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr7-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
182994 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr7-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937495.
182994 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 62361 variants from chromosome 7, leaving 120633.
Pruning complete.  62361 of 182994 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr7-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr7-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr8-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr8-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.
162061 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr8-filtered-set-2014Apr15_pruned_snps.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.93764.
162061 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Pruned 56678 variants from chromosome 8, leaving 105383.
Pruning complete.  56678 of 162061 variants removed.
Writing...
Marker lists written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr8-filtered-set-2014Apr15_pruned_snps.prune.in
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chr8-filtered-set-2014Apr15_pruned_snps.prune.out
.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chru-filtered-set-2014Apr15_pruned_snps.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chru-filtered-set-2014Apr15
  --indep-pairwise 10 10 0.5
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/chru-filtered-set-2014Apr15_pruned_snps

15488 MB RAM detected; reserving 7744 MB for main workspace.

Error: Invalid chromosome code 'scaffold2170' on line 1 of .bim file.
(Use --allow-extra-chr to force it to be accepted.)

Again, this will result in a substantially reduced set of SNPs. Below, we will filter each genotype file and concatenate the results into one merged plink file.

Review the chunk below, and then run (no changes necessary). IMPORTANT Review the snp_map output, and make sure that the genotypes and prune.in files map correctly - otherwise you will get nonsense later!

print(snp_map)

$`chr1-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr1-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr2-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr2-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr3-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr3-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr4-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr4-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr5-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr5-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr6-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr6-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr7-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr7-filtered-set-2014Apr15_pruned_snps.prune.in"

$`chr8-filtered-set-2014Apr15`
[1] "/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr8-filtered-set-2014Apr15_pruned_snps.prune.in"

If the snp_map is correct above, review the cell below and run.

for (genotype in names(snp_map)) {
    
    tmp <- sprintf("%s%s",output_directory, genotype)
    
    idx <- which(names(snp_map) == genotype)
    fileparts <- strsplit(genotype_files[[idx]], "/")[[1]]
    filename <- strsplit(fileparts[length(fileparts)], "\\.")[[1]][1]
    
    filter_cmd <- sprintf("
    
        %s --bfile %s \\
           --extract %s \\
           --make-bed \\
           --out %s
    ", plink_, 
       sprintf("%s%s", genotype_directory, filename), 
       snp_map[[genotype]],
       sprintf("%s%s_ld_filtered", output_directory, filename))
    
    system(filter_cmd)
    
}

PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr1-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
188757 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 122523 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.93859.
122523 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr1-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr2-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
169284 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 109028 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.938203.
109028 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr2-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr3-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
203743 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 135544 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937448.
135544 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr3-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr4-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
207603 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 135017 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937824.
135017 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr4-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr5-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
167564 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 108342 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937295.
108342 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr5-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr6-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
129085 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 84948 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.935851.
84948 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr6-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr7-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
182994 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 120633 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937881.
120633 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr7-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.
PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15
  --extract /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure//chr8-filtered-set-2014Apr15_pruned_snps.prune.in
  --make-bed
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered

15488 MB RAM detected; reserving 7744 MB for main workspace.
162061 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered.nosex
.
--extract: 105383 variants remaining.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.93787.
105383 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/chr8-filtered-set-2014Apr15_ld_filtered.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.

Finally, we will merge the filtered sets. Review the cell below and run (no changes required). (this is ugly code - sorry.)

combine_cmd <- sprintf("
    
    %s --bfile %s \\
       --merge-list %s  \\
       --make-bed \\
       --out %s
", plink_,
   first_file,
   sprintf("%sfiltered_files.txt", working_directory),
   sprintf("%smerged_genotypes", output_directory)
)
system(combine_cmd)

PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes.log.
Options in effect:
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files//chr1-filtered-set-2014Apr15_ld_filtered
  --make-bed
  --merge-list /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/filtered_files.txt
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes

15488 MB RAM detected; reserving 7744 MB for main workspace.
Performing single-pass merge (262 people, 921418 variants).

Pass 1: fileset #1 complete.
Pass 1: fileset #2 complete.
Pass 1: fileset #3 complete.
Pass 1: fileset #4 complete.
Pass 1: fileset #5 complete.
Pass 1: fileset #6 complete.
Pass 1: fileset #7 complete.
                                              
Merged fileset written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes-merge.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes-merge.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes-merge.fam
.
921418 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes.nosex
.
Using 1 thread (no multithreaded calculations invoked.
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937684.
921418 variants and 262 people pass filters and QC.
Note: No phenotypes present.
--make-bed to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes.bed
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes.bim
+
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes.fam
... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99%done.

Population structure

Now we get to the good parts. Commonly, principal components analysis (PCA) or multidimensional scaling (MDS) are used to determine population structure. MDS requires us to first calculate genetic distances (via identity-by-descent (IBD)). IBD calculations take forever. PCA however, can be calculated relatively quickly directly from the genotypes. Everything I have read suggests that PCA and MDS can be used interchangeably for approximating population structure. So to keep things efficient, we will use PCA.

Review the cell below and run (no changes required)

cmd <- sprintf("
               
    %s --bfile %s  \\
       --allow-extra-chr \\
       --pca %s  \\
       --out %s
", plink_, 
   sprintf("%smerged_genotypes", output_directory), 
   parameters$pop_struct_dimensions,
   sprintf("%spopulation_structure", output_directory)
)
system(cmd)

PLINK v1.90b3.31 64-bit (3 Feb 2016)       https://www.cog-genomics.org/plink2
(C) 2005-2016 Shaun Purcell, Christopher Chang   GNU General Public License v3
Logging to /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/population_structure.log.
Options in effect:
  --allow-extra-chr
  --bfile /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/merged_genotypes
  --out /mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/population_structure
  --pca 10

15488 MB RAM detected; reserving 7744 MB for main workspace.
921418 variants loaded from .bim file.
262 people (0 males, 0 females, 262 ambiguous) loaded from .fam.
Ambiguous sex IDs written to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/population_structure.nosex
.
Using up to 4 threads (change this with --threads).
Before main variant filters, 262 founders and 0 nonfounders present.
Calculating allele frequencies... 0%1%2%3%4%5%6%7%8%9%10%11%12%13%14%15%16%17%18%19%20%21%22%23%24%25%26%27%28%29%30%31%32%33%34%35%36%37%38%39%40%41%42%43%44%45%46%47%48%49%50%51%52%53%54%55%56%57%58%59%60%61%62%63%64%65%66%67%68%69%70%71%72%73%74%75%76%77%78%79%80%81%82%83%84%85%86%87%88%89%90%91%92%93%94%95%96%97%98%99% done.
Total genotyping rate is 0.937684.
921418 variants and 262 people pass filters and QC.
Note: No phenotypes present.

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Relationship matrix calculation complete.
[extracting eigenvalues and eigenvectors]
--pca: Results saved to
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/population_structure.eigenval
and
/mnt/DataDrive/MedicagoFiles/Analyses/PopulationStructure/plink_genotype_files/population_structure.eigenvec
.

This should produce 2 output files, population_structure.eigenval and population_structure.eigenvec (note, there will also be a log file). The eigenval file contains the eigenvalues for each of the prinicipal components, and describes the amount of variation attritbuted to each component. The eigenvec file contains the tranformed gentoypes (i.e. the principal components). As far as I know, plink does not output the rotation matrix.

Scree plot

Plot of the eigenvalues against the factor (component) number.

working_dir <- "/mnt/DataDrive/MedicagoFiles/Analyses/"
setwd(working_dir)

var_values <- read.csv("Medicago_Pop_Structure.eigenval", header = F)[, 1]
plot(var_values, xlab = "Factor", ylab = "Eigenvalue")
lines(var_values)

Looking at this, there is an elbow around 5 components. We’d probably get away with using this many quite nicely.

Metaplot

Here, we will plot the first two principal components and colour by various attributes that we know about the data, specifically: country of origin and flowering date

working_dir <- "/mnt/DataDrive/MedicagoFiles/Analyses/"
setwd(working_dir)
library(data.table)
library(ggplot2)

pca <- fread("Medicago_Pop_Structure.eigenvec")
meta <- fread("Pipeline/Traits/MedicagoTraits.combined")
country <- fread("../AccessionCountries.csv")

vis_data <- merge(pca[, 1:4, with = F], meta, by.x = c("V1", "V2"), by.y = c("FID", "IID"), all.x = TRUE)
vis_data <- merge(vis_data, country, by.x = "V2", by.y = "Accession", all.x = TRUE)
head(vis_data)


png("~/Documents/GitHub/GWASPipeline/Sandpit/PopulationStructure/Medicago_Population_Structure.png", height = 1000, width = 1000)
ggplot(vis_data, aes(x = V3, y = V4)) +
    geom_point(aes(colour = Country), alpha = 0.5, size = 5) +
    geom_text(aes(label = Country), colour = "darkblue", alpha = 0.6) +
    guides(colour = "none") +
    theme_minimal() +
    theme(axis.text = element_blank()) + xlab("") + ylab("") + ggtitle("Population Structure")
dev.off()

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